Tay-Sachs disease is an autosomal recessive disorder caused by mutations in the alpha-chain polypeptide of the A form of beta- hexosaminidase, a lysosomal enzyme composed of two chains (alpha,beta). Such lesions result in a spectrum of disease states ranging from severe to mild. Although the disorder is in general rare, Ashkenazi Jews, have a 10-fold higher gene frequency then the general population for a severe form of the disorder known as "classic" Tay-Sachs disease. It has been assumed that only one mutation gives rise to the severe form of Tay-Sachs disease in this ethnic group. We previously found that Ashkenazi Jewish patients with class Tay- Sachs disease lacked mRNA coding for the alpha-chain of beta- hexosaminidase, but retained a grossly intact alpha-chain gene indicating the presence of a subtle defect. During the past year we have completed sequence analysis of the promoter region, exon and splice junction regions and polyadenylation signal area of the mutant gene. Only one difference was observed between these sequences: at the 5' boundary of intron 12 a guanosine in the conserved splice junction dinucleotide sequence GT had been altered to a cytidine. The alteration is presumed to be functionally significant and to result in aberrant mRNA splicing. We developed an assay which utilized the polymerase chain reaction to amplify the region encompassing the lesion to screen patients and carriers for the splice junction mutation. In two Ashkenazi patients tested only one allele harbored the splice junction mutation, others were negative for this mutation, and 30% of the obligate heterozygote carriers tested displayed the mutation. These results demonstrate that at least two lesions underlie the classic form of Tay-Sachs disease in the Ashkenazi Jewish population. We have isolated alpha-chain genomic clones form an Ashkenazi Jewish patient negative for the splice junction lesion and are sequencing critical region of this gene to determine this second mutation.